Revised Abstract: DESCRIPTION (provided by applicant): Polarized cell migration (PCM) is key for cell defense against infections, wound healing, axonal growth, embryonic development, tumor cell metastasis and angiogenesis. Our hypothesis postulates that P-REX1, an exchange factor for Rac, establishes critical protein-protein interactions with receptors, G proteins and novel elements participating in PCM. Our specific aims are: 1) To define molecular links leading to the activation of Rac by G proteins. 2) To determine if P-REX1, identified as a Rac activator responding to Gbetagamma and PI3K (Welch et al, 2002, Cell 108:809), establishes protein interactions relevant for PCM. 3) To determine the role of G betagamma- and P-REXl-interacting proteins in the activity of Rac and PCM. Our studies consider the modular architecture exhibited by P-REX1, which contains two DEP and two PDZ domains, suggestive of modulation by direct protein-protein interactions. Our long term goal is to understand the molecular aspects of signal transduction required for PCM of endothelial cells and to identify molecular antiangiogenic tools. The role of Gbetagamma in PCM will be monitored in the presence of phosducin-like protein- and GRK2- derived peptides. P-REX1 deletion mutants lacking the different structural domains will be prepared to identify the structural domains in P-REX1 recognized by Gbetagamma and PI3K and to reveal their role in the activation of Rac and PCM. To identify novel elements able to modulate endothelial cell migration, Gbetagamma- and P-REXl interacting proteins will be cloned by yeast two hybrid and, in parallel, will be identified by proteomic approaches. It will be determined if CXCR1 and CXCR2 GPCRs containing PDZ-interacting motives in their carboxyl terminal domain are able to establish stable interactions with P-REX1. CXCR4 that does not contain that motif will be used as a control. Human HEK293T cells will be the model to study molecular interactions. Those cells will be transfected with the diverse DNA constructs. Recombinant molecules will be expressed fused to GST, EGFP, Myc or HA tags that will facilitate their study. Endothelial cells will be used to determine the role of Gbetagamma, P-REX1 and their interacting proteins, on the activation of Rac and polarized migration responding to the activation of angiogenic G protein coupled receptors. Discovery of molecular elements that might impede unwanted endothelial cell migration will provide elements for the design of antiangiogenic treatments.